Given our interest in the APC/C and its downstream targets, we have been focusing our studies on an indirect target of the APC/C. Separase is the protease that cleaves the cohesin complex that holds homologs together at meiosis. Securin inhibits separase from carrying out this role until the metaphase to anaphase transition, at which time securin is ubiquitinated by the APC/C and degraded by the 26S proteasome.[unreadable] [unreadable] We have taken a genetic approach to identify regulators and substrates of separase. We have three mutant alleles of sep-1 and have carried out a suppression screen with one temperature-sensitive alleles of sep-1. We have identified three suppressors that restore viability to sep-1 mutants at the non-permissive temperature. One of these mutants is an intragenic suppressor; the other two are extragenic. We have recently determined that one of these suppressor mutations is in a phosphatase gene called pph-5. This phosphatase mutant, in an otherwise wild-type background, has no obvious phenotypes on its own. RNAi of this pph-5 suppresses the embryonic lethality of sep-1 alleles as does a deletion allele of this gene. To further understand how PPH-5 works in the separase pathway, we have undertaken a proteomics approach and have generated transgenic animals that express a TAP-tagged version of PPH-5. Purification of the tagged phosphatase and its associated proteins, followed by mass spectrometry, has revealed two interacting proteins. One of these interacting proteins is DAF-21, a C. elegans HSP90 ortholog. RNAi of daf-21 has previously been shown to cause sterility in C. elegans, a phenotype similar to that of RNAi of wee-1.3, an inhibitor of CDK-1. Our current model is that PPH-5 influences the activity of DAF-21, which in turn regulates WEE-1.3 and CDK-1. Since CDK-1 is a known regulator of SEP-1 activity, it may be the indirect mechanism by which pph-5 mutations suppress the embryonic lethality of sep-1 mutants. Alternatively, PPH-5 is also thought to function in the RAS/RAF pathway and we have some genetic data to suggest that our pph-5 mutant can enhance a ras gain-of-function allele. We are pursuing this analysis to fully understand the mechanism of suppression by pph-5.